The major goal of this project is to develop and exploit a tractable, preclinical mouse models of metastatic melanoma that recapitulate the clinical progression of surgical removal of primary tumors, treatment with adjuvant therapy and clinical recurrence at distant sites. We see a translational opportunity to devise a novel strategy to prevent the clinical recurrence of metastatic disease. The models will be designed to incorporate normal immune system function to maintain an appropriate tumor microenvironment and provide superior tumor-host interactions, an approach that is significantly more likely to yield biologically and clinically relevant data. Since metastasis often targets internal organs and the timing of its recurrence may vary greatly, the preclinical model will allow for the non-invasive, long-term monitoring of disease progression within the immunocompetent mouse. To meet these criteria, we originally developed as a proof-of-concept a syngeneic immunocompetent mouse model of Lewis Lung Carcinoma (LLC) using tissue that was never adapted to cell culture and labeled using a high-titer lentivirus encoding a luciferase/GFP fusion reporter protein, developed by this laboratory in collaboration with Dom Esposito at the Protein Expression Laboratory, Advanced Technology Program (Day et al., Pig Cell Melanoma Res 22:283-295, 2009). Our decision to employ the NSCLC mouse LLC model was well aligned with the new NCI Lung Cancer Program, and provided a robust, well-characterized system to validate our preclinical approach. LLC exhibits highly favorable growth kinetics and virtually 100% take rate of lung metastases in C57BL/6 mice, and was instrumental in the successful development of early conventional chemotherapies that are still used in the clinical today. For this preclinical model we employed an in vivo-maintained LLC stock that had never been adapted to cell culture, an approach that had been reported to yield more reliable preclinical data. In full collaboration with Dr. Melinda Hollingshead (DTP, NCI), we have established a stably labeled bank of highly metastatic LLC tissue (LLC-Luc/GFP) through multiple rounds of subcutaneous transplantation, resection, and selection of Luc/GFP+ metastatic clones in syngeneic C57BL/6 mice. Our preclinical approach was to subcutaneously inoculate Luc/GFP-labeled LLC cells into albino C57BL/6 mice and resect at a selected tumor size, at which time drug treatment was initiated. Recurrence of clinically significant LLC macrometastases was evident in the lungs as Luc/GFP+ lesions within a week, and entire drug studies were completed in a month. The BL signal allowed the monitoring of disease after resection of the primary tumor, the progression of metastatic growth over time, and the response of the tumor to therapy. We have found that this resection/recurrence metastasis model fulfilled multiple requirements for a tractable and robust syngeneic preclinical model of advanced stage disease, one that we anticipate may more accurately predict therapeutic response. We treated mice in a setting akin to post-surgical first-line adjuvant chemotherapy using cisplatin, paclitaxel and/or antiangiogenic agents. As in the clinic these drugs were found to be most effective against progression when used in combination. However, the response of metastases to agents could not be predicted from, and often opposed, their effects on subcutaneous tumors. Moreover, time to macrometastasis onset, rather than growth, correlated with both mouse survival and treatment efficacy. This work has been published (Day et al., Int. J. Cancer 130:190-9, 2011). We have noted that even low expression of the xenobiotic reporters GFP and/or luciferase can render growth of labeled metastatic tumors unpredictable in syngeneic mice and severely limit the utility of any immunocompetent preclinical model. We have therefore developed transgenic mice in both FVB/N and C57BL/6 backgrounds that express the Luc/GFP fusion gene at an irrelevant site (in this case the anterior pituitary through use of a rat growth hormone promoter) (Day et al., PLoS One. 2014 Nov 4;9(11):e109956). These glowing head mice are thus pre-tolerized to both foreign markers, and we note that clearly labeled metastatic lesions arise much more regularly, consistently, and with a stronger signal in the lungs of inoculated, resected host mice. These mice are currently being employed in our ever improving preclinical models (Day et al., PLoS One, 2014). The glowing head mice have already been sent to multiple investigators and are now being distributed by Jackson Laboratories. The primary goal now is to combine all the tools and reagents we have developed over the last few years to study the mechanisms by which BRAF mutant, NRAS mutant, and wildtype BRAF/NRAS mutant melanomas recur at metastatic sites when patients fail targeted and immunotherapeutic drug treatment. This extensive preclinical effort is being performed in full collaboration with Dr. Shyam Sharan and CAPR, and in collaboration with Drs. Nick Restifo (CCR), Marcus Bosenberg (Yale), Martin McMahon (UCSF), Jedd Wolchok (MSKCC) and Jennifer Wargo (MD Anderson). We have already developed a wildtype BRAF/NRAS metastatic melanoma model using our UV-initiated HGF transgenic mouse. Metastatic BRAF mutant melanomas have now been generated and are being fully characterized and incorporated into our studies. Preclinical trials with immunotherapy (e.g., anti-CTLA-4, anti-PD-1, anti-PD-L1) and targeted therapy (e.g., BRAFi, MEKi, METi) are already underway and providing novel insights into why some melanomas but not others respond to immune checkpoint inhibitors. We have written a PRIMER for Cell that reviews optimal use of preclinical cancer models (Day, Merlino, Van Dyke, CELL, 2015). We have new data suggesting that neoantigens generated by UV exposure and other sources may be associated with the responses of melanoma tumors to immune checkpoint inhibitors. In vivo screens are underway to identify the neoantigens that are responsible. Moreover, anti-PD-L1 antibody treatment has shown good activity in our mouse model, and the responses resemble those seen in the clinic with melanoma patients.